An enzyme-linked immunosorbent assay (ELISA) was employed to determine the L. intracellularis-specific IgA and IgG immunoglobulin response, as described previously [23 (link)]. Briefly, L. intracellularis bacteria were heated for 5 min at 95°C and centrifuged at 1.2 × 104 g for 60 sec. The supernatant was discarded and the pellet was resuspended in PBS. The wells of ELISA plates (Greiner, Germany) were coated with 100 μL of heat-killed L. intracellularis bacteria (4 mg/mL) in 100 mM sodium carbonate (pH 9.7). The L. intracellularis-specific IgA was detected using serum from control and test mice and HRP-conjugated goat-anti-mouse antibodies (Southern Biotech) and measured using an automated ELISA spectrophotometer (Tecan, Austria) at 492 nm.
Automated elisa spectrophotometer
Manufactured by Tecan
Sourced in Austria
The Automated ELISA spectrophotometer is a laboratory instrument designed to perform enzyme-linked immunosorbent assay (ELISA) analysis. It is capable of automatically measuring the absorbance of ELISA plate samples at specific wavelengths, which is a core function in the quantitative analysis of various analytes, such as proteins, hormones, and antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti mouse antibody, by Southern Biotech (1 mentions)
Elisa plate, by Greiner (1 mentions)
Nunc maxisorp 96 well plate, by Thermo Fisher Scientific (1 mentions)
O phenylenediamine, by Merck Group (1 mentions)
Hrp conjugated goat anti mouse igg and igm, by Southern Biotech (1 mentions)
96 well high binding polystyrene plates, by Greiner (1 mentions)
O phenylenediamine dihydrochloride opd, by Merck Group (1 mentions)
3 protocols using automated elisa spectrophotometer
1
ELISA for L. intracellularis IgA/IgG
2
Quantifying Mucosal and Systemic Antibody Levels
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We determined immunoglobulin A (IgA) levels in the vaginal wash and systemic immunoglobulin G (IgG) levels in the serum of immunized mice (n = 5) using ELISA with Nunc Maxisorp 96-well plates (Nunc, Denmark). The proteins were coated (500 ng/well) overnight at 4oC in carbonate-bicarbonate buffer (pH = 9.6). The plate was washed thrice with 0.05% PBS with Tween 20 and blocked with 5% skim milk powder at 37oC for 2 h. After washing, wells containing serum (1:100) or vaginal wash (1:2) were added and incubated at 37oC for 1 h. Goat anti-mouse conjugates (IgG or IgA) at 1:5,000 dilutions were added, and after incubation, the color was developed using O-phenylenediamine as a substrate (Sigma-Aldrich, USA). Colorimetric changes were measured at 492 nm using an automated ELISA spectrophotometer (Tecan, Austria). The standard curve describing the relation between the concentration of standards and their absorbance value was generated. The antibody concentration for each sample was expressed as the mean optical density at 492 nm ± standard error of the mean.
3
Quantifying Antigen-Specific Antibody Levels
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Antigen-specific immunoglobulin (Ig) G and IgM levels were measured by indirect ELISA using serum samples collected on weeks 0, 1, 2, 4, 6, and 8 post-primary vaccination. Briefly, purified antigens were coated (500 ng/well) on 96-well high-binding polystyrene plates (Greiner Bio-One, Kremsmuster, Austria) and incubated at 4 °C overnight. The following day, wells were blocked with 5% skim milk (BD Difco) for 1 h at room temperature and washed three times with 0.1% phosphate-buffered saline with tween 20 (PBSt). Next, wells were incubated with serum samples (1:50) for 1 h at 37 °C, and plates were washed three times with PBSt. HRP-conjugated goat anti-mouse IgG and IgM (Southern biotech, AL, USA) secondary antibodies at 1:3000 dilution were added, and the plates were incubated at 37 °C for 1 h. After washing three times with PBSt, Color development was achieved with the prepared O-phenylenediamine dihydrochloride (OPD, Sigma-Aldrich) substrate in phosphate-citrate buffer containing H2O2 at room temperature in the dark. Optical density (OD) was measured at 492 nm using an automated ELISA spectrophotometer (Tecan, Groedig, Austria).
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